Nitric oxide (NO) has diverse roles both in normal and pathological processes including the regulation of blood pressure, in neurotransmission, and in the macrophage defense systems (Snyder, S. H., et al., Scientific American, May 1992, 68). NO is synthesized by three isoforms of nitric oxide synthase (NOS), two of which, one in endothelial cells (eNOS) and one in neuronal cells (nNOS), are constitutive, and the one, in macrophage cells, which is inducible (iNOS). These enzymes are homodimeric proteins that catalyzed a five-electron oxidation of L-arginine, yielding NO and citrulline. The role of NO produced by each of the NOS isoforms is quite unique. Overstimulation or overproduction of individual NOS isoforms plays a role in several disorders including septic shock, arthritis, diabetes, ischemia-reperfusion injury, pain and various neurodegenerative diseases (Kerwin, J. F. Jr., et al., J. Med. Chem. 1995, 38, 4343). For example, the role of NO in cerebral ischemia can be protective or destructive depending on the stage of evolution of the ischemic process and on the cellular compartment producing NO (Dalkara, T., et al. Brain Pathology, 1994, 4, 49). While the NO produced by eNOS is likely beneficial by acting as a vasodilator to improve blood flow to the affected area (Huang, Z., et al. J. Cereb. Blood Flow Metab. 1996, 16, 981), NO produced by nNOS may contribute to the initial metabolic deterioration of the ischemic penumbra, resulting in larger infarcts (Hara, H., et al., J. Cereb. Blood Flow Metab. 1996, 16, 605). The metabolic derangement that occurs during ischemia and subsequent reperfusion results in the expression and release of several cytokines that activate iNOS in several cell types including some of the central nervous system. NO can be produced at cytotoxic levels by iNOS, and increased levels of iNOS contribute to progressive tissue damage in the penumbra, leading to larger infarcts (Parmentier, S., et al. Br. J. Pharmacol., 1999, 127, 546). Inhibition of i-NOS has been shown to ameliorate cerebral ischemic damage in rats (Am. J. Physiol., 268, R286 1995).
NO produced by i-NOS is also thought to play a role in diseases that involve systemic hypotension such as toxic shock and therapy with certain cytokines. It has been shown that cancer patients treated with cytokines such as interleukin 1 (IL-1), interleukin 2 (IL-2) or tumor necrosis factor (TNF) suffer cytokine-induced shock and hypotension due to NO produced from macrophages, i.e., inducible NOS (i-NOS) (Chemical & Engineering News, Dec. 20, 33, 1993). i-NOS inhibitors can reverse this. Suppression of adjuvant induced arthritis by selective inhibition of i-NOS is reported in Eur. J. Pharmacol., 273, p. 15–24 (1995).
n-NOS inhibition has also been shown to be effective in antinociception, as evidenced by activity in the late phase of the formalin-induced hindpaw licking and acetic acid-induced abdominal constriction assays (Br. J. Pharmacol., 110, 219–224, 1993). Also, opioid withdrawal in rodents has been reported to be reduced by n-NOS inhibition (see Neuropsychopharmacol., 13, 269–293, 1995).
Neuropathic pain, as defined by the International Association for the Study of Pain (IASP), is pain initiated or caused by a primary lesion or dysfunction in the nervous system and may be associated with either the central or peripheral nervous system. Neuropathic pain can be subcategorized into central and peripheral neuropathic pain corresponding to lesions or dysfunction to the peripheral and central nervous system respectively. In contrast to acute nociceptive pain, which is finite, localized, subsides with healing or removal of the noxious substance, and serves a protective biological function by minimizing the exposure potential of continuing tissue damage, chronic neuropathic pain serves no protective biological function. Thus rather than being a symptom of a disease process it is itself a disease process that can persist long after the initial injury. If chronic pain is inadequately treated, associated symptoms of chronic anxiety, depression, fear, sleeplessness and social impairment may result.
Nociceptive and neuropathic pain are caused by different neurophysiological processes and thus tend to respond to different treatments. Nociceptive pain, whether somatic or visceral in nature, is mediated by A-delta and C-fibres located in skin, bone, vicera etc. Examples of this type of pain include post-operative pain, pain associated with trauma and arthritic pain and can be effectively managed with opioids or NSAIDS. Neuropathic pain is caused by pathological changes such as peripheral or central neuronal sensitization, abonormal somatic and sympathetic interactions, and central sensitization related to damaged inhibitory neuronal function. These abnormal states are manifested in the development of hyperalgesia and allodynia. Hyperalgesia corresponds to augmented pain intensity in response to a normally painful stimulus while allodynia refers to nociceptive response to normally innocuous stimuli, whether due to tactile (touch) or cold. There are many causes of neuropathic pain giving rise to hyperalgesia and allodynia and include major and minor surgery (eg dental surgery, mastectomy), major and minor trauma (eg. spinal cord damage, sports related injuries), loss of limbs (eg. phantom limb pain) neurological disorders (stroke, MS, fibromyalgia), psychiatric and affective disorders, chemically induced injury (eg chemotherapy such as cisplatin and taxol treatment), metabolic disorders (eg. diabetes), viral induced pain (eg. Shingles, HIV associated neuropathy, postherpetic neuraligia from herpes zoster) and mechanical or tactile allodynia associated with migraine. Any disease which can result in nerve damage can give rise to neuropathic pain states.
Early evidence for the role of NO in pain transmission (Meller and Gebhart; Pain, 1993, 52, 127–136) has lead to intensive research into its involvement. Extensive work has shown that nitric oxide synthase is rich in laminae I-III of the dorsal horn of the spinal cord (Dun. et. al. Neuroscience 1993; 54: 845–857), an area known to be involved in pain processing (Willis and Westlund. J. Clin. Neurophysiol. 1997; 14: 2–31) and that NOS is often co-localized with inhibitory GABA-ergic inhibitory neurons (Lian et. al. Neuroscience 1994; 61: 123–132). Physiological studies have support the role of NO in spinal modulation of pain transmission (Przesmycki et. al. Eur. Neuropsycholpharmacol. 1999; 9: 115–121) but it appears to be more involved in thermal evoked pain or changes in sustained nociceptive responses following injury rather than acute nociceptive reflexes (Meller et. al. Eur. J. Pharmacol. 1992; 214: 93–96, Meller and Gebhart; Pain 1993; 52: 127–136, Stanfa et. al. Brain Res. 1996; 737: 92–98). Recent studies using direct electrochemical measurements of NO in laminae I-III have shown that NO is released only after a titanic burst percutaneous stimulation, characteristic of activation of activation of Aβ and Aδ fibres, but not after single shock low intensity stimulus (Schulte and Millar. Pain 2003; 103:139–150). NO synthesis could be nearly abolished with the NOS inhibitor L-NAME without effect on receptive fields. Burst stimuli are known to produce windup (frequency dependent increase in the excitability of spinal cord neurones) in the dorsal horn which can be attenuated with the application of NOS inhibitors. Activation of C-fibres also produced a rise in NO levels.
Considerable evidence has demonstrated the NO and NOS are involved in the central mechanisms of inflammatory thermal hyperalgesia at the level of the spinal cord (Tao et. al. Neuroscience; 2000; 95: 525–533, Eur. J. Pharmacol. 2000; 392: 141–145, Neuroscience 2002; 112: 439–446, Handy and More Br. J. Pharmacol. 1998; 123:1119–1126, Neuropharmacology, 1998, 37: 37–43). It appears that both spinal nNOS and iNOS mRNA are upregulated during peripheral inflammation (Guhring et. al. J. Neurosci. 2000; 20: 6714–6720, Wu et. al. Exp. Brain. Res. 1998; 118: 457–465, Pain 2001; 94: 47–57) while eNOS expression after carrageenan injection is not (Tao et. al. Neurosci. 2003; 120: 847–854). Studies have shown that nNOS appears to the predominant player in inflammatory hyperalgesia. For instance, the time course and intensity of carrageenan-induced thermal hyperalgesia in iNOS knockout and wild type mice are similar in both early and late phase (secondary component). In addition, intrathecal (spinal) administration of neuronal selective nNOS inhibitor 7-nitroindazole but not eNOS selective L-N-(1-iminoethyl)ornithine, significantly reduced carrageenan-induced thermal hyperalgesia in iNOS knockout mice. It appears that nNOS may compensate for iNOS function and that iNOS is likely sufficient but not essential for late phase of inflammatory mediated thermal hyperalgesia (Tao et. al. 2003; 120: 847–854).
The use of NOS inhibitors in the treatment of disease has been described, for example, in international patent application nos. WO 94/12163, WO 93/13066, WO 94/12165, WO 95/00505, WO 95/09619, WO 95/09621, WO 95/10266, WO 95/11231, WO 95/11014, WO 96/01817 and WO 98/50382, and in European patent application nos. EP 446699, EP 547558, and EP 558468.
NOS inhibitors can be therapeutic in many disorders, but preservation of physiologically important nitric oxide synthase function requires the development of isoform-selective inhibitors.